Methods for determining a protein-protein interaction can be roughly categorized into two groups. One is a method characterized by using a protein having been separated from living cells. Examples of such a method include surface plasmon resonance, protein mass spectroscopy, and anisotropy measurements. However, these methods have difficulty determining an interaction in an environment similar to an actual intracellular environment.
Then, as the second method, a method has been developed, in which a protein-protein interaction is determined using living cells. Typical methods thereof are a yeast two hybrid system which detects a transcriptional activity of a reporter, and modified methods thereof. Besides, another method has been also developed which utilizes reconstitution of enzymes such as β-galactosidase and dihydrofolate reductase (DHFR).
Nevertheless, these methods have problems that they are incapable of determining a position where a protein-protein interaction has taken place (positional information on the protein-protein interaction), as well as a period until a protein-protein interaction takes place, a period until the interaction ends, a duration of the interaction, and the like (temporal information on the protein-protein interaction).
Meanwhile, the method for determining a protein-protein interaction using living cells also includes a method utilizing reconstitution of a fluorescent protein. Nevertheless, once reconstituted, the fluorescent protein does not dissociate. Accordingly, this method has a problem that it is incapable of determining a period until a protein-protein interaction ends, a duration of the interaction, and the like. Further, there is another problem that a period until a protein-protein interaction takes place and the like cannot be determined because emission of fluorescence requires a certain time after a protein-protein interaction takes place.
On the other hand, as a method for determining a protein-protein interaction in living cells, fluorescence resonance energy transfer (FRET) has been developed which detects energy transfer dependent on a distance between molecules. This method has an advantage of obtaining positional information and temporal information on where and when a protein-protein interaction takes place. Nevertheless, since a positional relation between a donor fluorescent protein and an acceptor fluorescent protein used in the method is important to determine the protein-protein interaction, the method involves a complicated step of investigating the optimization of a linker (spacer) connecting these fluorescent proteins to a detection-target protein, so that such a system has been difficult to construct. Further, it has also been difficult to analyze the result due to cross excitation by which an acceptor fluorescent protein is excited, and due to bleed-through in which fluorescence of a donor fluorescent protein bleeds through a filter (absorption filter) set for detecting fluorescence of an acceptor fluorescent protein. Moreover, use of fluorescent proteins of two colors (donor fluorescent protein and acceptor fluorescent protein) also brings about a problem that only a limited number of fluorescent proteins are usable in order to obtain information other than a detection-target protein.
Recently, Tobias Meyer et al. have reported a method for determining a protein-protein interaction by utilizing intracellular localization (PTL 1). In this method, one of proteins subjected to interaction determination is fused to a protein that specifically binds to a particular site in a cell, while the other of the proteins subjected to interaction determination is fused to a fluorescent protein or the like. Then, these fusion proteins are expressed in a cell, and the protein-protein interaction is determined on the basis of a signal of the fluorescent protein or the like at the particular site in the cell.
In addition, Nibert et al. have reported a method for determining a protein-protein interaction by using a fusion protein in which one of proteins subjected to interaction determination is fused to a protein for forming a viral inclusion body, and using, as an indicator, accumulation of the other of the proteins subjected to interaction determination in the viral inclusion body (PTL 2).
However, in these methods for determining a protein-protein interaction by utilizing intracellular localization, one of proteins subjected to interaction determination is forcibly (artificially) translocated and confined at a particular site in a cell. Accordingly, the determination is impossible at a site where a protein-protein interaction naturally takes place, that is, in an intracellular environment unique to the protein-protein interaction, which brings about a problem that positional information on the protein-protein interaction cannot be obtained, and other similar problems. Moreover, it is also impossible to determine the interaction between proteins localized in a natural state at the same site as the site of the intracellular localization.
Against this problem, Sara Peterson Bjorn et al. have reported a method for determining a protein-protein interaction (redistribution-trap method) in which proteins are allowed to interact with each other in an intracellular environment where the proteins naturally function, and then the cells are stimulated with a drug or the like to thereby induce aggregate formation from the interacting proteins, the aggregate formation being indicative of the interaction (PTL 3).
However, this method needs to stimulate cells at certain time so that the aggregate formation can be induced, and also needs to remove the drug or the like used for the stimulation to determine the presence or absence of an interaction subsequently after the stimulation. Hence, the method has problems such as being incapable of obtaining temporal information on when the protein-protein interaction takes place, and incapable of determining a protein-protein interaction that changes (takes place, ends, takes place again, and so forth) for a certain period and at a certain position.